Abi3500 capillary sequencer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ABI3500 capillary sequencer is a DNA sequencing instrument manufactured by Thermo Fisher Scientific. It uses the Sanger sequencing method to determine the nucleotide sequence of DNA samples. The instrument utilizes capillary electrophoresis technology to separate and detect fluorescently labeled DNA fragments.

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14 protocols using abi3500 capillary sequencer

Variant Validation and Classification

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All variants were classified according to the guidelines from the American College of Medical Genetics to pathogenic, likely pathogenic, of uncertain significance, likely benign or benign [14 (link)], and novel variants were submitted to the ClinVar Database (https://www.ncbi.nlm.nih.gov/clinvar/). Pathogenic and likely pathogenic variants were classified as disease causing variants.
Candidate variants found by NGS were validated using Sanger sequencing if the coverage at the variant site of the exome sequencing result was below 30x and/or the base quality score below 500, in accordance with previously published recommendations [15 (link)]. Furthermore, Sanger sequencing was employed to resolve cases with a suspected compound heterozygous combination of variants and for other familial segregation analyses. Sequencing was carried out using BigDye 3.1 sequencing chemistry (Life Technologies), followed by capillary electrophoresis on the ABI 3500 capillary sequencer (Life Technologies).
Primer sequences are available upon request.

Likar T., Hasanhodžić M., Teran N., Maver A., Peterlin B, & Writzl K. (2018). Diagnostic outcomes of exome sequencing in patients with syndromic or non-syndromic hearing loss. PLoS ONE, 13(1), e0188578.

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Genotyping Mouse Tail DNA

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Mouse tail DNA for initial genotyping of pups was isolated using DirectPCR Lysis Reagent (Tail) (Viagen Biotech, Los Angeles, CA) containing proteinase K (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. PCRs were performed with GoTaq Green Master Mix (Promega, Madison, WI) by using the following primers: R122C forward: 5’-GGAGAGTTTTCTCGCAGGTTC-3’, reverse: 5’-GTACCTGAAGAAGCCTCCAGC-3’; tdTomato forward: 5’-CTGTTCCTGTACGGCATGG-3’, reverse: 5’-GGCATTAAAGCAGCGTATCC-3’; and Iqgap3-intact forward: 5’-CAGCTGCAGTATGAGGGTGT-3’, reverse: 5’-GGTAATGGAGAAGCGCAGCAGCC-3’.
For genotyping by Sanger sequencing, PCR products were amplified using TaKaRa LA Taq DNA Polymerase (Takara, Kusatsu, Japan), were treated with ExoSAP-IT (USB Corporation, Cleveland, OH), and then labeled with the BigDye Terminator Cycle Sequencing Kit v3.1 (Life Technologies, Carlsbad, CA) using the same PCR primers as in the amplification reactions. Sequencing products were loaded on an ABI3500 capillary sequencer (Life Technologies). The following primers were used for sequencing: R122C_seq forward: 5’-TGATGGCCGGCAATGATGAGAACTACTCCG-3’, reverse: 5’-GCGGCCGCAGCACCGGAGACTTCAGAAGTTGCTAAACC-3’.

Douchi D., Yamamura A., Matsuo J., Lee J.W., Nuttonmanit N., Melissa Lim Y.H., Suda K., Shimura M., Chen S., Pang S., Kohu K., Kaneko M., Kiyonari H., Kaneda A., Yoshida H., Taniuchi I., Osato M., Yang H., Unno M., Bok-Yan So J., Yeoh K.G., Huey Chuang L.S., Bae S.C, & Ito Y. (2022). A Point Mutation R122C in RUNX3 Promotes the Expansion of Isthmus Stem Cells and Inhibits Their Differentiation in the Stomach. Cellular and Molecular Gastroenterology and Hepatology, 13(5), 1317-1345.

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HAV Genome Amplification and Sequencing

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Amplification of the VP1‐P2B region (481 nucleotides [nt]) of HAV genome was performed with PrimeScript II High‐Fidelity One Step RT‐PCR Kit (Takara Bio) using previously reported primers.¹² After agarose gel purification with QIAquick Gel Extraction Kit (Qiagen), the PCR products were processed with the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and analysed with a ABI3500 capillary sequencer (Thermo Fisher Scientific) to obtain sequence data. Sequenced fragments were assembled using CLC Main Workbench 8 software (Qiagen) and consensus sequences were extracted. The genotypes of HAV strains detected in this study were determined by BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Abe H., Ushijima Y., Bikangui R., Ondo G.N., Zadeh V.R., Pemba C.M., Mpingabo P.I., Igasaki Y., de Vries S.G., Grobusch M.P., Loembe M.M., Agnandji S.T., Lell B, & Yasuda J. (2020). First evidence for continuous circulation of hepatitis A virus subgenotype IIA in Central Africa. Journal of Viral Hepatitis, 27(11), 1234-1242.

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Viral Genome Sequencing Protocol

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To determine HRV types, the amplification of the VP4/VP2 (541 nucleotides) region of the viral genome was performed for HRV-positive samples using the PrimeScript II High-Fidelity One Step RT-PCR Kit (Takara Bio, Shiga, Japan) with previously reported primers [24 (link)]. After agarose gel purification with the QIAquick Gel Extraction Kit (Qiagen), PCR products were processed with the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and analyzed using an ABI3500 capillary sequencer (Thermo Fisher Scientific) to obtain sequence data. Sequenced fragments were assembled manually using Bioedit software v7.2.5 (https://thalljiscience.github.io/). The genotypes of the detected strains were determined using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). RT-PCR for EV- and HCoV OC43-positive samples or PCR- for HAdV-positive samples were performed using the following primers: EV [25 (link)], HCoV OC43 [26 (link)], and HAdV [27 (link)], although it was difficult to amplify the viral genomes. Regarding PIVs, we did not attempt PCR on positive samples because their Ct values were >38.

Ondo G.N., Ushijima Y., Abe H., Mahmoudou S., Bikangui R., Nkoma A.M., Mbadinga M.J., More A., Agbanrin M.D., Pemba C.M., Beh Mba R., Akim A.A., Lell B, & Yasuda J. (2024). Genetic Diversity and Detection of Respiratory Viruses Excluding SARS-CoV-2 during the COVID-19 Pandemic in Gabon, 2020–2021. Viruses, 16(5), 698.

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Viral Screening Protocol for Bunyavirales

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Virus screening was performed using a PrimeScript II High Fidelity One Step RT-PCR kit (Takara Bio, Shiga, Japan). We initially targeted partially conserved nucleotide sequences of three viral genera, namely Mammarenavirus, Orthohantavirus and Orthonairovirus, all belonging to the order Bunyavirales. For reverse-transcription (RT) PCR detection of Old World mammarenaviruses and orthohantaviruses, we used previously described pan-viral family primer sets [11, 22] . For orthonairoviruses, a primer set was designed based on the alignment of the sequences of a highly conserved region in the L gene (Table S1 available in the online version of this article). RT-PCR was performed under the following conditions: 10 min at 45 °C, 2 min at 94 °C and 35 cycles of 10 s at 98 °C, 15 s at 45 °C and 10 s at 68 °C. After purification from the agarose gel using the QIAquick Gel Extraction kit (QIAGEN, Hilden, Germany), the PCR products were processed using the BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific) and analysed with an ABI 3500 capillary sequencer (Thermo Fisher Scientific) for Sanger sequencing. The obtained sequences were identified using blast searches (https://blast.ncbi.nlm.nih.gov). The sequences of the RT-PCR amplicons were deposited in the DNA Data Bank of Japan (DDBJ) database under the following accession numbers: LC706368-LC706436.

Ozeki T., Abe H., Ushijima Y., Nze-Nkogue C., Akomo-Okoue E.F., Ella G.W.E., Koumba L.B.M., Nso B.C.B.B., Mintsa-Nguema R., Makouloutou-Nzassi P., Makanga B.K., Nguelet F.L.M., Ondo G.N., Mbadinga M.J.V.M., Igasaki Y., Okada S., Hirano M., Yoshii K., Lell B., Bonney L.C., Hewson R., Kurosaki Y, & Yasuda J. (2022). Identification of novel orthonairoviruses from rodents and shrews in Gabon, Central Africa. The Journal of general virology, 103(10).

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Multiplex Microsatellite Genotyping of Elms

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The total genomic DNA was isolated from approximately 20 mg of leaf tissue using an ISOLATE II PLANT DNA kit (Bioline, London, UK). Suitable markers originally described for Ulmus species were tested for their ability to provide repeatable, high-quality results, sufficient polymorphism and unambiguous allele binding. Finally, six polymorphic markers: Ulm19, Ulm2, Ulm3, Ulm6, Ulm9 (Whiteley et al. 2003 (link)), and UR188a (Zalapa et al. 2008 (link)) were simultaneously amplified in a multiplex reaction using Multiplex Master Mix (Qiagen, Hilden, Germany). The polymerase chain reaction (PCR) program was as follows: 3 min at 94 °C; 30 cycles of 15 s at 94 °C, 90 s at 53 °C, and 2 min 72 °C, and 20 min at 72 °C. The fluorescently labeled PCR products, along with a size standard (GeneScan 600 LIZ, Thermo Fisher Scientific, Waltham, Massachusetts, USA), were separated on an ABI 3500 capillary sequencer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Alleles were identified based on their size using GeneMapper software (ver. 5.0; Thermo Fisher Scientific, Waltham, Massachusetts, USA), and all variants were checked and approved manually (Litkowiec 2022 ).

Litkowiec M., Chudzińska M., Pasławska A., Pałucka M., Kozioł C, & Lewandowski A. (2022). Population history, genetic variation, and conservation status of European white elm (Ulmus laevis Pall.) in Poland. Annals of Forest Science, 79(1), 38.

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16S rRNA Gene Amplification and Analysis

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The partial 16S rRNA gene was amplified from DNA extractions by PCR using fluorescently labeled primers for bacteria and archaea (Additional file 1: Table S4). The DNA Polymerase KAPA2G Robust HotStart ReadyMix (Sigma-Aldrich, Missouri, United States) was used within a suitable master-mix according to manufacturer’s instructions. PCR amplifications were carried out on a T100 Thermocycler (Bio-Rad Laboratories, Inc., Hercules, California, United States). Products of the PCR were end-treated for the correction of the overhanging ends effect [37 (link)] and were cleaned with a Millipore MultiScreen PCR_μ96 filter plate (Merck KGaA, Darmstadt, Germany). Finally, the products were resuspended in 25 μL ddH₂O. Purified PCR amplicons were digested by using the restriction enzyme AluI according to the manufacturer’s instructions. Each 1 μL of digestion product was mixed with 18.65 μL Hi-Di formamide and 0.35 μL GeneScan LIZ 600 Size Standard (Thermofisher Scientific™, Massachusetts, United States), denatured and analyzed using ABI 3500 capillary sequencer (Thermofisher Scientific™).

Schmautz Z., Espinal C.A., Bohny A.M., Rezzonico F., Junge R., Frossard E, & Smits T.H. (2021). Environmental parameters and microbial community profiles as indication towards microbial activities and diversity in aquaponic system compartments. BMC Microbiology, 21, 12.

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Molecular Identification of SFG Rickettsiae

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Ticks, morphologically identified according to Manilla (1998 ) and Iori et al. (2005 ), were submitted to DNA extraction using a commercial kit (QIAamp DNA mini Kit, Qiagen, Hilden, Germany). PCR protocols targeting gltA, ompA, and ompB genes were carried out to reveal the presence of SFG rickettsiae (Scarpulla et al. 2016 (link)). Amplicons were gel purified for downstream analysis consisting of DNA classical sequencing (Big Dye terminators, v3.1, chemistry and ABI3500 capillary sequencer, Applied Biosystems). GltA and ompB sequences, of 351 and 396 bp, respectively, were analyzed (Geneious software, Biomatters Ltd.) and finally challenged in GenBank using the nBLAST algorithm.

Scarpulla M., Barlozzari G., Salvato L., De Liberato C., Lorenzetti R, & Macrì G. (2018). Rickettsia helvetica in Human-Parasitizing and Free-Living Ixodes ricinus from Urban and Wild Green Areas in the Metropolitan City of Rome, Italy. Vector Borne and Zoonotic Diseases, 18(8), 404-407.

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Genetic Variability Analysis of Sorbus

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Analysis of the genetic variability of Sorbus species was carried out using 13 nuclear microsatellite markers (nSSR—nuclear Simple Sequence Repeats) (Gianfranceschi et al. 1998 ; Oddou-Muratorio et al. 2001 ; Liebhard et al. 2002 ; Kamm et al. 2009 (link); González-González et al. 2010 ). Primer sequences, PCR conditions and multiplex PCR protocols used in the analysis are listed in Supporting Information—Table S1. PCR products were separated on the ABI 3500 capillary sequencer (Applied Biosystems) and the length of each DNA fragment was sized relative to an internal size standard and calculated using GeneMapper software. Each allele peak designation was checked and confirmed manually. Any inconsistent samples were repeated to ensure the observed allele sizes were not artefacts or scoring errors.

Hebda A., Kempf M., Wachowiak W., Pluciński B., Kauzal P, & Zwijacz-Kozica T. (2020). Hybridization and introgression of native and foreign Sorbus tree species in unique environments of protected mountainous areas. AoB Plants, 13(1), plaa070.

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Targeted Sequencing Workflow for Variant Identification

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Target enrichment was performed using in-solution technology (NimbleGen SeqCap EZ Library v.3.0, Roche), and the resulting target libraries were sequenced by Illumina sequencing technology (HiSeq2000). Raw image files were processed by Illumina basecalling software (CASAVA 1.7) using default parameters. Paired-end reads were aligned to the human genome (UCSC GRCh37/hg19) with the Burrows-Wheeler aligner (BWA v. 0.7.10) [38 (link)]. Presumed PCR duplicates were removed using Picard's MarkDuplicates. The Genome Analysis Toolkit (GATK 3.3) [39 (link)] was used for realignment of sequences encompassing indels and for base quality recalibration. Somatic single-nucleotide variants were detected using Mutect software v.1.1.6 [40 (link)] and small indels were identified through a comparison between indels called in individual C-CSC lines and their matched nontumoral samples by means of the GATK Haplotype Caller algorithm [41 (link)], as previously described [42 (link), 43 (link)]. The resulting SNVs and small indels were annotated by SnpEff v3.6 [44 (link)] and dbNSFP2.8 [45 (link)] in terms of functional impact of variants [46 (link), 47 ]. Variant validation and genotyping were performed by direct sequencing using the ABI BigDye Terminator Sequencing kit (Applied Biosystems) and an ABI3500 capillary sequencer (Applied Biosystems).

Verdina A., Di Rocco G., Virdia I., Monteonofrio L., Gatti V., Policicchio E., Bruselles A., Tartaglia M, & Soddu S. (2017). HIPK2-T566 autophosphorylation diversely contributes to UV- and doxorubicin-induced HIPK2 activation. Oncotarget, 8(10), 16744-16754.

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